The discovery and better characterization of stem cells in foetal and adult tissues over the last decade has opened novel possibilities for cell therapy. These primitive or incompletely differentiated “progenitor cells”, which present high self-renewal capacity, can give rise to fully differentiated cells, or acquire specific differentiated phenotypes once administered to patients. Progenitor cells can be isolated from embryos or adult issues including peripheral blood, bone marrow and adipose tissue for instance, as well as other specialized tissues such as umbilical cord blood.
The increasingly well defined methods for the identification and isolation of progenitor cell populations have strongly encouraged the development of stem cell-based therapy strategies to be deployed clinically. Besides, as a means to circumvent ethical difficulties associated with the use of human foetal tissues, the isolation and transplantation of progenitor cells originating from adult tissues is favoured.
Regenerative cell therapy with progenitor cells seems particularly relevant for diseases in which organs are compromised in such a way that tissue reconstruction is required, e.g. to restore the function of a diseased organ, or when physiological repair mechanisms are impaired.
Thus, one notable field of application of progenitor cell therapy relates to cardiovascular diseases. In these cases, the primary goal is to enhance tissue perfusion by promoting the growth or emergence of a new vascular tree, or to improve the function of existing vessels. Other examples of applications of progenitor cell therapy also include the revascularization and repair of defective heart tissues with vascular progenitors.
In most instances, the progenitor cells of interest are delivered into the general circulation. Current approaches thus rely mostly on the ability of the transplanted cells to directly exert their effects in the circulation, or to find their way to the target tissue, integrate therein and exert their beneficial effects through innate and little understood mechanisms.
In order to maximize the therapeutic effects, local administration of the progenitor cells has also been attempted. It is then hoped that a greater proportion of the transplanted progenitor cells will engraft in the tissue, which would provide enhanced beneficial effects. However, the transplanted cells often fail to adhere to the local substratum, and rapidly clear out of the receiving tissue through mechanisms which remain mostly enigmatic, but seem to include apoptosis.
Thus, the lack of adhesive capacity of the engrafted cells, in particular the lack of specific adhesive capacity for the targeted tissues, may compromise cell engraftment into the targeted tissue and lead to significant loss of the administered cells (Aicher et al. (2003) Circulation 107:2134-9). Administered cells may thus be either destroyed in situ through innate immune response pathways, pass into the blood flow, and get cleared out by organs such as the liver and spleen, or get trapped into lung capillaries, for instance.
Moreover, cell therapy is met with further limitations beyond the engraftment of administered cells into the targeted tissue. The engrafted cells may be ineffective at participating to the reconstruction of the targeted tissue. In particular, the failure of engrafted cells may be due to reduced mid-term and long-term survival, or to the loss of some of their properties, such as their angiogenic and/or vasculogenic activity, e.g. reduced growth factor and cytokine secretion, or their impaired capacity to differentiate and acquire the desired phenotype.
Besides the above-mentioned barriers to cell therapy, other difficulties arise from the pathologies which afflict the individual from whom the cells for engraftment have been isolated. For instance, it is known that circulating Endothelial Progenitor Cells (EPCs) originating from diabetic, obese, atherosclerotic, hypertensive, or smoking patients are less adhesive than those from healthy subjects, resulting in defective angiogenic and vasculogenic properties of these cells (Werner and Nickenig (2006) Arterioscler. Thromb. Vasc. Biol. 26:257-266; Werner and Nickenig (2006) J. Cell. Mol. Med. 10:318-332; Callaghan et al. (2005). Antioxid. Redox Signal 7:1476-1482; Loomans et al. (2005) Antioxid. Redox Signal 7:1468-1475; Roberts et al. (2005) J. Cell. Mol. Med. 9:583-591).
It has previously been proposed that treating EPCs preparations with growth factors prior to transplantation might enhance their pro-angiogenic effects. Thus, it has been shown that the adipose tissue-derived cytokine leptin could enhance significantly adhesion of EPCs to vitronectin-coated culture plates and to mature endothelial cells (Schroeter et al. (2006) 114:121). However, such a treatment has not been assessed in vivo.
As such, it is an object of the present invention to improve existing methods for preparing cells to be engrafted.
Blood coagulation, and arterial thrombosis in particular, involves platelet aggregation. An essential function of the aggregated platelets is to degranulate and release their contents into the blood stream. Platelet α-granules contain Thrombospondin-1 (TSP1), which forms up to 25% of released platelet proteins. TSP1 promotes enhanced platelet aggregation by participating in the formation of platelet-platelet, platelet-endothelial cell and platelet-extracellular matrix bridges, and further participates in inflammatory reactions involving monocytes-macrophages, lymphocytes, neutrophils, basophils, and even fibroblasts.
TSP1 was initially identified as a platelet released protein following thrombin activation. TSP1 can also be expressed in the vascular wall by smooth muscle cells after mechanical injury or during diabetes, and by endothelial cells during thrombosis, after perturbations of laminar blood flow, or consecutive to hypoxia.
TSP1 has a unique and complex structure with multiple domains, that activate a number of specific extracellular receptors. Different domains can thus sometimes induce apparently opposite signal pathways in the single cell type, as detailed below.
TSP1 interacts mainly with the scavenger receptor CD36 at the cell surface, as well as with Integrin-Associated Protein (CD47/IAP) and integrins.
TSP1 is mainly known as the archetypal endogenous anti-angiogenic factor. TSP1 plays an anti-angiogenic function in tumours and blocks their progression. TSP1 also inhibits re-endothelialisation following vascular injury and retinal neo-vascularisation in vivo.
The anti-angiogenic effects of TSP1 are mediated by at least one of its Type I domains and CD36 receptor activation. Indeed TSP1 binds CD36 through its CSVTCG sequence of its type I domains (Guo et al., (1997) Cancer Res, 57:1735-1742). In microvascular endothelial cells and in tumor endothelial cells, TSP1 binding to CD36 induces caspase activation, p38-MAPK and p59-Fyn kinase phosphorylation, resulting in apoptosis (Jimenez et al (2000) Nature Medicine 6:41-48). TSP1 is also known to trigger fibroblast apoptosis (Graf et al. (2002) Apoptosis 7:493-498), an additional cue as to how TSP1 may compromise tissue repair and vascularization.
Another well known TSP1 receptor is the quasi-ubiquitous Integrin-Associated Protein (CD47/IAP). TSP1 binding to CD47/IAP occurs through the carboxyterminus of TSP1 which notably comprises the sequence RFYVVMWK (4N1-1, SEQ ID NO: 3).
A peptide from the C-terminal domain of thrombospondin-1 known as 4N1-1 (Frazier (1993) J. Biol. Chem. 268:8808-14) or its derivative 4N1K can bind to CD47/IAP in a similar manner as TSP1. This CD47/IAP-binding domain is highly conserved between species and TSP isoforms.
The activation of CD47 by TSP1 and 4N1-1 has been shown to induce the expression of CAM family proteins in mature endothelial cells. TSP1 thus enables the recruitment of circulating cells, in particular α4β1 integrin-expressing inflammatory cells, towards the CAM-expressing vascular endothelium (Narizhneva et al. (2005) FASEB J. 19:1158-60).
However, such mechanisms have not been described in circulating undifferentiated progenitor cells, Furthermore, it has been shown that the activation of CD47 by TSP1 or 4N1-1 also induces apoptosis and the demise of mature endothelial cells (Freyberg et al. (2000) Biochem Biophys Res Commun 271:584-588; Freyberg et al. (2001) Biochem Biophys Res Commun 286:141-149; Graf et al. (2003) Apoptosis 8:531-538). CD47 agonists are thus known anti-angiogenic agents, that could be used in novel potential approaches to prevent the vascularization and growth of solid tumors (Manna et al. (2004) Cancer Res 64:1026-1036). CD47 expression and activation has also been linked to the inhibition of angiogenesis and endothelial death in models of angiogenesis in vivo (Isenberg et al. (2007) Circ. Res. 100:712-20).
It has further been proposed that TSP1 leads to the dismantlement of the intracellular skeleton and focal adhesion plaques, inhibition of proliferation, caspase activation, and eventually to apoptosis. Moreover, TSP1 inhibits Nitric Oxide (NO) signals, a powerful promoter of normal endothelial cell function and survival (Isenberg et al. (2006) J. Biol. Chem. 281:26069-80; Isenberg et al. (2007) J. Biol. Chem. 282:15404-15). Endothelial apoptosis thus appears to be a major aspect of the anti-angiogenic effects of TSP1.
In addition, TSP1 can be expressed by progenitor cells derived from diabetic bone marrow (Li et al (2006) Circ Res 98:697-704). In this case TSP1 was shown to block their adhesion and their contribution to vascular re-endothelialization after vascular injury, in keeping with its anti-angiogenic role in mature endothelial cells.
Accordingly, in view of prior studies, TSP1 and CD47 agonists do not seem to be usable to increase adherence of cells, and therefore to promote pro-angiogenic therapy or neo-vascularisation.
Besides, it is particularly interesting to note that systemic injection of significant doses of 4N1K peptide is not lethal and does not modulate gross coagulation parameters (Bonnefoy et al. (2006) Blood 107:955-64), despite its known prothrombotic effects on platelet aggregation (Voit et al. (2003) FEBS Lett. 544:240-5). It thus seems unlikely that 4N1-1, 4N1K or derived peptides have significant toxic effects in healthy subjects.